Advancing Label-Free Imaging Through CARS Microscopy: From Signal Formation to Biological Interpretation.

Label-free imaging is becoming ever more important, especially in modern molecular biophysics. This method allows observation of biological structures and dynamics without the alteration caused by dyes or genetic labels. Coherent Anti-Stokes Raman Scattering (CARS) microscopy represents a unique method that utilizes the intrinsic vibrational signatures of biomolecules, thereby transforming the field. Fluorescence-based methods show marked sensitivity, but may cause photobleaching, labeling artifacts, and inadequate biochemical detection. CARS enables chemically specific, real-time imaging of molecular structures, e.g., lipids, proteins and nucleic acids, within their natural environment. Over the past decade, advances in laser technology, detection methods, and computer analysis have turned CARS from a rare optical phenomenon into a useful tool applied in many fields, from basic research on molecular structure to practical biomedical imaging. This review presents the principles of CARS microscopy and the latest achievements in this field, highlighting its impact on molecular and cellular biophysics, as well as exploring the potential of artificial intelligence and multimodal approaches to increase its applications in precision medicine. In this context, CARS serves both a state-of-the-art imaging technique and a means of transforming internal molecular vibrations into information useful in biology and biophysics. In this way, it combines the physical sciences with molecular biology, enabling innovative biomedical research.